Biodiversity Data Journal 1: e1013 OO)
doi: 10.3897/BDJ.1.e1013
open access
Taxonomic paper
Eupolybothrus cavernicolus Komericki & Stoev
sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae):
the first eukaryotic species description combining
transcriptomic, DNA barcoding and micro-CT
imaging data
Pavel Stoevt+, Ana Komeriéki8, Nesrine Akkari!, Shanlin Liu, Xin Zhou, Alexander M. Weigand*'§,
Jeroen Hostens!t, Christopher |. Hunter#+, Scott C. Edmunds*+, David Porco8§, Marzio Zapparolil!,
Teodor Georgievt, Daniel Mietchen™4+, David Roberts**, Sarah FaulwettertT 444, Vincent Smith**,
Lyubomir PenevSs§
t+ National Museum of Natural History, Sofia, Bulgaria
+ Pensoft Publishers, Sofia, Bulgaria
§ Croatian Biospeleological Society, Zagreb, Croatia
| Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark
4 China National GeneBank, BGI-Shenzhen, Shenzhen, China
# Goethe-University, Institute for Ecology, Evolution and Diversity, Frankfurt am Main, Germany
tt Bruker microCT, Kontich, Belgium
++ GigaScience, BGI HK Ltd., Tai Po, Hong Kong, China
§§ Université de Rouen - Laboratoire ECODIV, Mont Saint Aignan Cedex, France
|| Universita degli Studi della Tuscia, Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Viterbo,
Italy
1 Museum fir Naturkunde — Leibniz-Institut fur Evolutions- und Biodiversitatsforschung, Berlin, Germany
## The Natural History Museum, London, United Kingdom
ttt National and Kapodestrian University of Athens, Athens, Greece
+++ Hellenic Centre for Marine Research, Heraklion, Greece
§§§ Institute of Biodiversity & Ecosystem Research - Bulgarian Academy of Sciences and Pensoft Publishers, Sofia, Bulgaria
Corresponding author: Pavel Stoev (projects@pensoft.net)
Academic editor: Robert Mesibov
Received: 19 Oct 2013 | Accepted: 23 Oct 2013 | Published: 28 Oct 2013
Citation: Stoev P, Komeri¢ki A, Akkari N, Liu S, Zhou X, Weigand A, Hostens J, Hunter C, Edmunds S, Porco D,
Zapparoli M, Georgiev T, Mietchen D, Roberts D, Faulwetter S, Smith V, Penev L (2013) Eupolybothrus
cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae): the first eukaryotic species
description combining transcriptomic, DNA barcoding and micro-CT imaging data. Biodiversity Data Journal 1:
e1013. doi: 10.3897/BDJ.1.e1013
ZooBank: urn:|sid:zoobank.org:pub:09D4F004-B7DD-4223-AFO0D-ED22E22EBOD0
© Stoev P et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
2 Stoev P et al.
Abstract
We demonstrate how a classical taxonomic description of a new species can be enhanced
by applying new generation molecular methods, and novel computing and imaging
technologies. A cave-dwelling centipede, Eupolybothrus cavernicolus Komericki & Stoev
sp.n. (Chilopoda: Lithobiomorpha: Lithobiidae), found in a remote karst region in Knin,
Croatia, is the first eukaryotic species for which, in addition to the traditional morphological
description, we provide a fully sequenced transcriptome, a DNA barcode, detailed
anatomical X-ray microtomography (micro-CT) scans, and a movie of the living specimen
to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a
new species for the first time, we create a high-resolution morphological and anatomical
dataset that allows virtual reconstructions of the specimen and subsequent interactive
manipulation to test the recently introduced ‘cybertype’ notion. In addition, the
transcriptome was recorded with a total of 67,785 scaffolds, having an average length of
812 bp and N50 of 1,448 bp (see GigaDB). Subsequent annotation of 22,866 scaffolds was
conducted by tracing homologs against current available databases, including Nr,
SwissProt and COG. This pilot project illustrates a workflow of producing, storing,
publishing and disseminating large data sets associated with a description of a new taxon.
All data have been deposited in publicly accessible repositories, such as GigaScience
GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses
used ensure their accessibility and re-usability.
Keywords
cybertaxonomy, gene sequence data, micro-CT, data integration, molecular systematics,
caves, Croatia, biospeleology
Introduction
While 13,494 new animal species were discovered by taxonomists in 2012 (index of
Organism Names), animal diversity on the planet continues to decline with unprecedented
speed (Balmford et al. 2003). Changes and intensification of land use, habitat destruction,
human population growth, pollution, exploitation of marine resources and climate change
are among the major factors that lead to biodiversity impoverishment, and for the first time
in human history, the rate of species extinction may exceed that of species discovery
(Wheeler et al. 2012). The rapid pace of extermination has forced taxonomists to speed up
the process of biodiversity investigation. The ‘turbo-taxonomy’ approach, combining
molecular data, concise morphological descriptions, and digital imaging, has recently been
introduced (Butcher et al. 2012, Riedel et al. 2013a) as one solution for the global loss of
taxonomic expertise, part of the problem generally referred to as ‘taxonomic impediment’
(Wagele et al. 2011). Accelerated ‘pipeline’ descriptions of 178 new species of parasitic
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 3
wasps (Butcher et al. 2012) and 101 new species of Trigonopterus weevils (Riedel et al.
2013b) were recently used to exemplify the concept.
Here, we present a more holistic approach to taxonomic descriptions. It is exemplified
through a new cave centipede, Eupolybothrus cavernicolus Komericki & Stoev sp. n.,
recently discovered by biospeleologists in Croatia. To the best of our knowledge, this is the
first time the description of a new eukaryotic species has been enhanced with rich genomic
and morphological data, including a fully sequenced transcriptome, DNA barcodes,
detailed X-ray micro-computed tomography scanning (micro-CT), and a video of a living
specimen showing behavioural features. In this increasingly data-driven era, a further aim
of this study is to set a new standard for handling, management and publishing of various
data types. It is essential that data are easily accessible to researchers in every field of
science, and able to be integrated from many sources, to tackle complex and novel
scientific hypotheses. Rapid advances and increasing throughput of technologies such as
phenotyping, genome-scale sequencing and meta-barcoding are now producing huge
volumes of data, but there has been a lag in efforts to curate, present, harmonise and
integrate these data to make them more accessible and re-usable for the community.
Furthermore, by employing micro-CT scanning we test for the first time in a new taxon the
recently introduced ‘cybertype’ notion (Faulwetter et al. 2013) of high-resolution virtual
morphological and anatomical data libraries allowing reconstruction and interactive
manipulation of type specimens.
To respond to the increasing interest in exposing and publishing biodiversity data (see e.g.,
Penev et al. 2011, Costello et al. 2013, Drew et al. 2013) and following the recent
developments in open access data publishing (Smith et al. 2013) we also propose a novel
workflow in the Biodiversity Data Journal of producing, storing, evaluating, publishing and
disseminating complex data sets. The large-scale data handling, management and storage
was provided by the GigaScience GigaDB database (see Stoev et al. 2013), with
transcriptomic and annotation data made publicly available to the most stringent metadata
standards in INSDC (NCBI/EMBL/DDBJ) databases, GigaDB and the relevant datatype
specific repositories.
The study group
The subfamily Ethopolyinae Chamberlin, 1915 is known to comprise some of the largest
lithobiomorphs in the world, with several species reaching 45-50 mm in length. At present,
the subfamily includes four more or less well defined genera: Bothropolys Wood, 1862 with
around 40 species from North America and East Asia; Archethopolys Chamberlin, 1925
with three species from the southwestern USA, Zygethopolys Chamberlin, 1925 with four
species from western Canada and the USA, and Eupolybothrus Verhoeff, 1907 with 23
valid and 15 doubtful species and subspecies assigned to seven subgenera ranging from
Southern Europe and North Africa to the Near and Middle East, including the largest
Mediterranean islands Corsica, Sardinia, Sicily, Crete and Cyprus (Zapparoli and
Edgecombe 2006, Zapparoli and Edgecombe 2011). The genus Eupolybothrus exhibits the
highest species diversity in the Italian and Balkan peninsulas (Zapparoli 2003), where a
4 Stoev P et al.
number of cave-dwelling species have restricted distribution ranges. A further 66 species-
level taxa proposed in Eupolybothrus are currently considered to be junior synonyms,
although their taxonomic status might change in the light of future taxonomic and molecular
studies. The exact placement of genus Ethopolys Chamberlin, 1912, with twelve species in
two subgenera from western Canada and the USA is uncertain, being treated in
contemporary literature as either a synonym of Bothropolys (Zapparoli and Edgecombe
2006, Zapparoli and Edgecombe 2011) or a valid genus (Mercurio 2010).
While some species of Eupolybothrus and the genus itself have been treated recently in
several publications (see e.g., Eason 1970, Zapparoli 1984, Zapparoli 1995, Zapparoli
1998, Zapparoli and Edgecombe 2006, lorio 2008, Stoev et al. 2010), the other three
genera, with few exceptions (e.g., Matic 1974, Ma et al. 2008, Ma et al. 2009, Ma 2012)
have remained out of the scope of contemporary studies. Nevertheless, it is also far from
being fully revised, as a number of problems are still in need of modern scrutiny. These
mainly concern: 1) a high number of vaguely described or/and poorly known species and
subspecies, mostly from the Balkans and Anatolia, known only from their original
description; 2) an outdated subgeneric classification that lacks any phylogenetic
framework; and 3) a high number of cryptic taxa in the E. nudicornis (Gervais, 1837), E.
litoralis (L. Koch, 1867) and E. tridentinus (Fanzago, 1874) species-groups, as recently
revealed by application of DNA barcoding (Porco et al. 2011, Komericki et al. 2012).
Further, Stoev et al. (2010) found high interspecific divergence values (20.8% mean value)
between two closely related Eupolybothrus species in another barcoding study with
mitochondrial Cytochrome C Oxidase subunit | (COI). Two other studies (Edgecombe and
Giribet 2003, Spelda et al. 2011) contributed genomic data by analysing DNA barcodes for
E. fasciatus and E. tridentinus from Italy and Germany, respectively. The present study is
part of an ongoing revision of the subfamily Ethopolyinae (Stoev et al. 2010, Porco et al.
2011, Komericki et al. 2012).
Materials and methods
Collected material and morphological study
The present study is based on eight specimens of Eupolybothrus cavernicolus Komericki &
Stoev sp.n. belonging to the Croatian Biospeleological Society (CBSS), the National
Museum of Natural History, Sofia (NMNHS) and the Natural History Museum of Denmark
(ZMUC). The specimens were preserved in ethanol (70 or 96%) or RNA/ater (Qiagen,
USA). The morphological study of the new species was performed at NMNHS and CBSS
with a Zeiss microscope. For scanning electron microscopy (performed at ZMUC), parts of
the specimens were cleaned by ultrasonification, transferred to 96% ethanol and then to
acetone, air-dried, mounted on adhesive electrical tape attached to aluminium stubs,
coated with platinum/palladium and studied in a JEOL JSM-6335F scanning electron
microscope. Images were edited in Adobe Lightroom 4.3 and Adobe Photoshop CS 5. All
morphological images have been deposited in Morphbank. Terminology for external
anatomy follows Bonato et al. (2010).
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 5
Molecular experiments and sequencing
DNA barcode sequencing
DNA extraction was conducted in the the Canadian Centre for DNA Barcoding, Guelph on
complete animals or part of the leg of the specimens preserved in 96% ethanol. Standard
protocols of the Canadian Centre for DNA Barcoding were used for both DNA extraction
and amplification. All specimen data are stored in the Barcode of Life Data System (BOLD)
online database and are available also in the dataset DS-EUPCAV (http://
dx.doi.org/10.5883/DS-EUPCAV), where they are linked to the respective Barcode Index
Numbers clusters. This dataset contains sequences from ten species: E. cavernicolus
Komericki & Stoev sp. n., E. leostygis (Verhoeff, 1899), E. obrovensis (Verhoeff, 1930), E.
grossipes (CL Koch, 1847), E. gloriastygis (Absolon, 1916), E. nudicornis, E. litoralis, E.
kahfi, E. transsylvanicus (Latzel, 1882) and E. tridentinus. In addition, all sequences were
registered in GenBank (accession numbers KF 715038-KF715064, HM065042-HM065044,
HQ941581-HQ941585, JN269950, JN269951, JQ350447, JQ350449), one sequence of E.
fasciatus (Newport, 1845) was recovered from GenBank (accession number AY214420).
Two sequences from two Lithobius species were included as outgroups: L. austriacus
(Verhoeff, 1937) (MYFAB442-11) and L. crassipes L. Koch, 1962 (MYFAB443-11). The final
dataset comprises 39 sequences. Molecular delimitation of species was achieved by the
implementation of the Automatic Barcoding Gap Discovery (ABGD) procedure as
described in Puillandre et al. (2012) and by the reversed Statistical Parsimony (SP)
approach as suggested by Hart and Sunday (2007). A Neighbor-Joining (NJ) tree was built
for visualization.
For the ABGD method, we tested various model combinations to cross-check the obtained
results: relative gap with (X) ranging from 0.05 to 1.5, minimal intraspecific distance (Pmin)
of 0.001 and maximal intraspecific distance (Pmax) ranging from 0.02 to 0.11. Pmin and
Pmax refer to the genetic distance area where the barcoding gap should be detected,
whereas X defines the width of the gap. Distance calculation was based on the Kimura-2-
parameter model and a transition/transversion ratio of 2.0. The method was performed in
100 steps. Statistical Parsimony networks for the delineation of species were reconstructed
on the basis of 95% statistical confidence (i.e. connection probability) using the program
TCS 1.21 (Clement et al. 2000). The NJ-topology was calculated in MEGA 5.0 (Tamura et
al. 2011) using the K2P-model under the pairwise-deletion option and 1000 bootstrap
replicates. Intra- and interspecific genetic K2P-distances were calculated in MEGA 5.0 as
well.
Transcriptome sequencing
One entire adult male specimen of Eupolybothrus cavernicolus Komericki & Stoev sp. n.
was crushed and preserved in liquid RNA/ater (Qiagen, USA) immediately after being
captured. To extract total RNA, TRIzol reagent (Invitrogen, USA) was used according to the
manufacturer’s instructions. Messenger RNA (mRNA) was isolated from total RNA using a
Dynabeads mRNA Purification Kit (Invitrogen, USA). The mRNA was fragmented and
6 Stoev P et al.
transcribed into first-strand CDNA using SuperScript™I| Reverse Transcriptase (Invitrogen,
USA) and N6 primer (IDT). RNase H (Invitrogen, USA) and DNA polymerase | (Invitrogen,
Shanghai China; New England BioLabs) were subsequently applied to synthesize the
second-strand of the cDNA. The double-stranded cDNA then underwent end-repair, a
single ‘A’ base addition, adapter ligation, and size selection, indexed and PCR amplified to
construct a library. The extracted cDNA was utilised for library construction with an insert
size of 250 bp. Finally, the library was sequenced on the Illumina HiSeq2000 sequencing
platform (Illumina, Inc., San Diego, California, USA) at BGl-Shenzhen using a 150bp pair-
end strategy to generate a total of 2.5 Gb raw reads. Illumina HCS1.5.15.1 + RTA1.13.48.0
were applied to generate a “bcl’” file which was then downloaded to local computers.
Secondly, the “bcl’ file was converted to qseq format using BclConverter-1.9.0-11-03-08.
Finally, we separated individual sample data from multiplexed machine runs based on the
specific barcode primer sequences, and converted the file format to fastq.
Micro-CT scanning
The micro-CT scanning of one adult female specimen was performed at Bruker microCT,
Kontich, Belgium, using a SkyScan 1172 system with the following settings: 40kV, 0.43°
rotation step, acquiring 839 projection images from 360° with a pixel size of 8um. Prior to
scanning, the sample was dehydrated in graded ethanol: 50%, 70%, 90%, 100%, for 2
hours in total, and then transferred to HMDS (hexamethyldisilasane) for 2 hours, and air
dried. Reconstruction was done with the SkyScan software NRecon, using a modified
Feldkamp algorithm, and adjusting for beam hardening and applying ring artefact
correction resulting in 3865 cross sections in .omp format, with image size 2000x2000
pixels. The video of 3D volume renderings was created with CTVox, using the flight
recorder function, and saved as an AVI (Audio Video Interface) file. The obtained data were
processed through a transfer function where the different voxels with different grey value
were (or weren't) made opaque and where the color was assigned to a certain grey value.
The image stack is stored in GigaDB (Stoev et al. 2013) under a Creative Commons CCO
public domain waiver. The only software used was CTVox, a viewing software, not analysis
software (although you could argue that viewing the images is also a way of analyzing
them).
Abbreviations
T — Tergite, TT — Tergites, Legs: L — left, R — right; Plectrotaxy table: Cx — coxa, Tr —
trochanter, Pf — prefemur, F — femur, T — tibia, a, m, p stand for spines in respectively,
anterior, medial and posterior position.
Taxon treatments
Eupolybothrus cavernicolus Komericki & Stoev, 2013, sp. n.
* ZooBank urn:lsid:zoobank.org:act:6F 9A6F3C-68 7A-436A-9497-70596584678C
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ...
SRA project accession http:/Awww.ebi.ac.uk/ena/data/view/ERP003841
ArrayExpress accession http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1859
GigaDB http://dx.doi.org/10.5524/100063
GenBank KF 715043
GenBank KF 715049
GenBank KF 715050
GenBank KF 715059
BOLD dataset http://dx.doi.org/10.5883/DS-EUPCAV
MorphBank 999021821 &tsn=true
MorphoSource http://morphosource.org/index.php/Detail/SpecimenDetail/Show/
specimen _id/514
Materials
Holotype:
a.
country: Croatia; stateProvince: Knin; locality: NP Krka, village Kistanje, Hydroelectric
power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude:
44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtoco!: hand collected under clay
sediment; eventDate: 9 February 2013; individualCount: 1; sex: male; lifeStage: adult;
recordedBy: M. Lukié; institutionCode: CBSS; collectionCode: CHP536
Paratypes:
a. country: Croatia; stateProvince: Knin; locality: NP Krka, village Kistanje, Hydroelectric
power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m; verbatimLatitude:
44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtoco!: hand collected under
lump of clay; eventDate: 18 October 2012; individualCount: 1; sex: female; lifeStage:
adult; recordedBy: A. Komericki; institutionCode: CBSS; collectionCode: CHP517
- country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje,
Hydroelectric power plant Miljacka, cave Miljacka Il; verbatimElevation: 115 m;
verbatimLatitude: 44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtocol:
hand collected; eventDate: 9 February 2013; individualCount: 1; sex: male; lifeStage:
adult; recordedBy: M. Lukic¢; institutionCode: BGI
- country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje,
Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m;
verbatimLatitude: 44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtocol:
hand collected; eventDate: 18 October 2012; individualCount: 1; sex: female; lifeStage:
adult; recordedBy: H. Cvitanovié & A. Komericki; institutionCode: NMNHS;
collectionCode: NUNHS-CHILOPODA-1/2013
- country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje,
Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m;
verbatimLatitude: 44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtocol:
hand collected; eventDate: 4 May 2010; individualCount: 1; sex: female; lifeStage: adult;
recordedBy: A. Kirin & A. Komericki; institutionCode: ZMUC; collectionCode:
zmuc00029439
- country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, cave
Miljacka IV (= Spilja kod mlina na Miljacki); verbatimElevation: 115 m; verbatimLatitude:
44°00'12.8"N; verbatimLongitude: 16°01'08.8"E; samplingProtocol: hand collected;
eventDate: 4 May 2010; individualCount: 1; sex: male; lifeStage: adult; recordedBy: M.
Lukic; institutionCode: ZMUC; collectionCode: zmuc00029440
- country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje,
Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m;
Stoev P et al.
verbatimLatitude: 44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtocol:
hand collected; eventDate: 4 May 2010; individualCount: 1; sex: female; lifeStage:
subadult; recordedBy: A. Kirin; institutionCode: CBSS; collectionCode: CHP420
g. country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje, cave
Miljacka IV (= Spilja kod mlina na Miljacki); verbatimElevation: 115 m; verbatimLatitude:
44°00'12.8"N; verbatimLongitude: 16°01'08.8"E; samplingProtoco!l: hand collected;
eventDate: 4 May 2010; individualCount: 1; sex: damaged female; lifeStage: subadult;
recordedBy: A. Komericki; institutionCode: CBSS; collectionCode: CHP416
h. country: Croatia; stateProvince: Knin; verbatimLocality: NP Krka, village Kistanje,
Hydroelectric power plant Miljacka, cave Miljacka II; verbatimElevation: 115 m;
verbatimLatitude: 44°00'01.1"N; verbatimLongitude: 16°00'58.5"E; samplingProtocol:
hand collected; eventDate: 21 October 2012; individualCount: 1; sex: damaged male;
lifeStage: adult; recordedBy: A. Komericki; institutionCode: CBSS; collectionCode:
CHP552
Description
Description of holotype: Body length: approx. 30 mm (measured from anterior
margin of cephalic plate to posterior margin of telson); leg 15 — 22.6 mm long, or 75%
length of body.
Color: uniformly yellow-brownish to chestnut, margins of cephalic plate slightly darker
than inner parts (Fig. 1).
Figure 1.
Habitus of Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype, ex situ.
Head: cephalic plate broader than long (4.0 x 3.6 mm, respectively), as wide as T1
(Fig. 2a); surface smooth, with several minute scattered pits, setae generally absent,
except for a few emerging from the marginal ridge (above ocelli) and on the median
sulcus. Cephalic median sulcus contributing to biconvex anterior margin, marginal ridge
with a median thickening; posterior margin straight or slightly concave; transverse
suture situated at about 1/3 of anterior edge; posterior limbs of transverse suture
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 9
visible, connecting basal antennal article with anterior part of ocellar area. Ocelli: 1+14
blackish, irregular in shape, in 3-4 rows, outermost first seriate ocellus largest, ocelli of
the middle two rows medium-sized, those of inferior row smallest (Fig. 2b).
TOmosvary’s organ: moderately large (as large as a medium ocellus), oval, situated
on subtriangular sclerotisation below the inferiormost row of seriate ocelli (Fig. 2b).
Clypeus: with a cluster of 25-30 setae situated on the apex and near the lateral margin
(Fig. 3a). Antennae: right antenna composed of 71 articles, left antenna damaged after
61° article; slightly surpassing posterior margin of T11 (right) or T9 (left) when folded
backwards, basal 2 articles enlarged, less pilose; posterior 30 articles visibly longer
than broad, ultimate article approx. 1.3 times longer than penultimate one (Fig. 3b).
Forcipules: coxosternite subpentagonal (Fig. 4a), shoulders almost absent (steep),
lateral margins straight; anterior margin set off as a rim by furrow; coxosternal teeth 8
+8, median diastema well-developed, V-shaped, steep and narrow, porodont arising
from a pit below the dental rim, situated lateral to the lateralmost tooth; base of
porodont thinner then adjacent tooth, coxosternite sparsely setose anteriorly; setae
moderately large, irregularly dispersed (Fig. 4b). Forcipular trochanteroprefemur, femur
and tibia and proximal part of forcipular tarsungulum with several setae. Distal part of
forcipular tarsungulum about 3 times longer than proximal part (Fig. 4a).
Figure 2.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: cephalic plate, dorsal view
b: ocelli and Témésvary’s organ. Abbreviations: ocellus (O) and Témésvary’s organ (7)
10
Stoev P et al.
Figure 3.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: clypeus, ventral view; most setae broken off
b: tip of antenna
Figure 4.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: forcipules, ventral view
b: close up of coxosternum, ventral view. Abbreviations: porodonts (po).
Tergites: T1 wider than long, subtrapeziform, wider anteriorly, posterior margin straight
or slightly emarginated, marginal ridge with a small median thickening; TT3 and 5 more
elongated than 11, posterior margin slightly emarginated medially, posterior angles
rounded; posterior angles of T4 rounded; posterior margin of T8 slightly emarginated
medially, angles rounded; TT6 and 7 with posterior angles abruptly rounded (Fig. 5a);
TTY, 11, 13 with well-developed posterior triangular projections (Fig. 5b); posterior
margin of TT10, 12, 14 slightly emarginated, posterior-most part densely setose;
intermediate tergite subpentagonal, posterior margin deeply emarginated, lateral edges
bent upwards, covered with setae; middle part of posterior third of tergite densely
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 11
covered with setae; laterally, on both sides of the central setose area there are two
specific seta-free regions (Fig. 6a, sfa). All tergites smooth, setae present only on their
lateral margins.
Figure 5.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: tergite 7, dorsal view
b: tergites 12-13, dorsal view
Figure 6.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: tergite 14 and intermediate tergite, posteriodorsal view. Abbreviations: seta-free areas (sfa).
b: pretarsus of leg 10, ventral view. Abbreviations: anterior accessory claw (a), posterior
accessory Claw (p).
12
Stoev P et al.
Legs: leg 15 longest; leg 14 approx. 25% longer than legs 1-12, leg 13 only slightly
longer than legs 1-12; pretarsus of legs 1-14 with expanded fundus, larger posterior
accessory claw (approx. 1/3™ of fundus) and a slightly thinner and shorter anterior
accessory claw (= spine, sensu Bonato et al. 2010) (Fig. 6b); pectinal (seriate) setae
missing on tarsi 1 and 2 of leg 15, present in one short row on tarsus 2 of leg 14, and in
one row on tarsus 1 and two rows on tarsus 2 of legs 1-13 (Fig. 7a); pretarsus of leg 15
without accessory spines (Fig. 7b). Length of podomeres of leg 15: coxa 1.5 mm,
prefemur 3.7 mm, femur 4.0 mm, tibia 5.2 mm, tarsus 1 5.0 mm, tarsus 2 3.0 mm,
pretarsus 0.25 mm. Prefemur of leg 15 with a large apically rounded proximal knob
(Fig. 8) protruding mediad, latter slightly bent dorsad and bearing a peculiar cluster of
long setae on tip (Fig. 9a); posterior edge with well defined circular protuberance at
mid-distance between spines a and p dorsally, covered with long setae (Fig. 9b), rest of
prefemur covered with sparse setae. Dorsal spine p on prefemur (but also in other
podomeres and other legs) with characteristic bi- and tripartite tip (Fig. 10a). Legs 1-14
without particular modifications. Coxal pores: generally round, arranged in 4-5
irregular rows, pores of inner rows largest, size decreasing outwards; pores separated
from each other by a distance more than, or equal to their own diameter; number of
pores on leg-pair 12: L-36/R-33, 13 L-41/R-44, 14 L-52/R-49, 15: L-39/R-34 (Fig. 10b).
Figure 7.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: tarsus 1, tarsus 2 and pretarsus of leg 10, lateral view. Abbreviations: pectinal setae (ps).
b: pretarsus of leg 15
Eupolybothrus cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 13
Figure 8.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: prefemur 15, mesoventral view. Abbreviations: prefemoral knob (pk), circular setose
protuberance (cp), cluster of setae (sc).
b: close up of the prefemoral knob, ventral view
Figure 9.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: close up of the clusp of setae on male prefemur 15
b: close up of the setose protuberance on male prefemur 15
14
Stoev P et al.
Figure 10.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype.
a: close up of the tip of prefemoral spine p
b: coxal pore pit, meso-ventral view
Sternites: all sternites smooth, subtrapeziform, with few sparse setae, mainly at lateral
margins; posterior margins straight.
Genitalia: posterior margin of male first genital sternite deeply concave, up to half of its
length, posterior margin densely covered with long setae, the rest of sternite sparsely
covered with shorter setae; gonopod small, hidden behind the edge of first genital
sternite, with 4-5 short setae (Fig. 11).
Figure 11.
Eupolybothrus cavernicolus Komericki & Stoev sp. n., male paratype. Genitalia, posterio-dorsal
view.
Plectrotaxy: as in Table 1.
Eupolybothrus cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 15
Table 1.
Plectrotaxy of E. cavernicolus Komericki & Stoev sp. n., male holotype.
Ventral Dorsal
Cx Tr Pf F T Cx Tr Pf F T
1 amp amp amp amp a-p a
2 amp amp amp amp a-p a-p
3 amp amp amp amp a-p a-p
4 amp amp amp amp a-p a-p
5 amp amp amp amp a-p a-p
6 amp amp amp amp a-p a-p
7 amp amp amp amp a-p a-p
8 amp amp amp amp a-p a-p
9 amp amp amp amp a-p a-p
10 amp amp amp amp a-p a-p
11 amp amp amp a amp a-p a-p
12 m amp amp amp a amp a-p a-p
13 m amp amp amp a amp a-p a-p
14 m amp am a a amp a-p a-p
15 am m amp am a a am p -
Description of male paratypes: All characters like in the holotype, except the
following: length of leg 15: prefemur 2.5 mm; femur 3.5 mm; tibia 4 mm; tarsus 1 3.7
mm; tarsus 2 2.5 mm; pretarsus 0.3 mm; ocelli: 1+12-1+13; antennae composed of
68-70 articles; coxosternal teeth: 6+7.
Description of adult female paratype: Body length: approx. 31 mm; leg 15 approx.
20-21 mm, or 68% length of body. Color: uniformly yellow-brownish to chestnut, head
and 11 darker, legs yellowish, margins of tergites darker; distal parts of tarsungulum,
coxosternal teeth and pretarsi of all legs dark brown to blackish.
Head: cephalic plate broader than long (3.9 x 3.5 mm, respectively), as wide as
anterior part of T1; surface smooth, with several pits scattered throughout the head and
16
Stoev P et al.
giving rise to trichoid setae. Cephalic median sulcus contributing to biconvex anterior
margin, marginal ridge with a median thickening; posterior margin slightly concave;
transverse suture situated at about 1/3 of anterior edge; posterior limbs of transverse
suture visible, connecting basal antennal article with anterior part of ocellar area.
Ocelli: 18 blackish, subequal in size, in 3-4 rows. T6ém6svary’s organ: moderately
large (as large as or slightly larger than a medium ocellus), oval, situated slightly above
the cephalic edge below the inferiormost row of ocelli. Clypeus: with a cluster of about
25 trichoid setae situated on the apex. Antennae: approx. 22 mm long, composed of
67 articles, reaching the middle of T10 when folded backwards, basal 2 articles
enlarged, less setose; posterior 30 articles visibly longer than broad, ultimate article
approx. 1.3 times longer than penultimate one. Forcipules: coxosternite
subpentagonal, shoulders almost absent, lateral margins straight; anterior margin set
off as a rim by furrow; coxosternal teeth 7+7, median diastema well-developed, V-
shaped, subparallel and narrow, porodont arising from a pit below the dental rim,
situated lateral to the lateralmost tooth; base of porodont thinner then adjacent tooth,
coxosternite sparsely setose anteriorly; setae moderately large, irregularly dispersed.
Medial side of forcipular trochanteroprefemur, femur and tibia and proximal part of
forcipular tarsungulum setose. Distal part of forcipular tarsungulum about 3 times
longer than proximal part.
Tergites: T1 wider than long, subtrapeziform wider anteriorly, posterior margin slightly
concave; TT3 and 5 more elongated than 11, posterior margin slightly concave
medially, posterior angles rounded; T2 almost entirely covered by 11, only
posteriormost part surpassing the margin of T1; posterior margin of TT4 and 6 straight,
posterior angles abruptly rounded; T7 rectangular, posterior margin straight, posterior
angles abruptly rounded; T8 approx. 1.4 times longer than T7, posterior margin of T8
slightly concave medially, angles abruptly rounded; TT9, 11, 13 with a well-developed
posterior triangular projections; TT10 and 12 subequal in size, approx. 1.2 times longer
than T8, posterior margin slightly emarginated; posterior margin of 114 slightly
emarginated, surface smooth, posterior-most part covered with just a few trichoid setae
(much more setose in male, see Fig. 6a); intermediate tergite subpentagonal, posterior
margin deeply emarginated, surface smooth, lateral edges bent upwards, a few trichoid
setae emerging from the posterior and lateral edges; areas covered with spines and
setae, as well as the specific setose free areas present in male (Fig. 6a, sfa) absent.
Legs: leg 15 longest, leg 14 latter approx. 25% longer than legs 1-12, leg 13 only
slightly longer than legs 1-12; pretarsus of legs 1-14 with a more expanded fundus,
larger posterior accessory claw (approx. 1/3™ of fundus) and a slightly thinner and
shorter anterior accessory claw (= spine, sensu Bonato et al. 2010); pectinal (seriate)
setae missing on tarsi 1 and 2 of leg 15, present in one short row on tarsus 2 of leg 14,
and in one row on tarsus 1 and two rows on tarsus 2 of legs 1-13; pretarsus of leg 15
without accessory spines. Leg 15 slender and elongate, without particular
modifications. Bifurcated spines present irregularly on most podomeres. Coxal pores:
generally round, forming 4-5 irregular rows, pores of inner rows largest, size
Eupolybothrus cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 17
decreasing outwards; pores separated from each other mostly by a distance more than
or equal to their own diameter.
Sternites: subtrapeziform in shape, anterior part wider; lateral sides straight in all but
ultimate sternite, where they are slightly convex; sternite surface smooth, shining,
covered with a few sparse setae, mainly at lateral margins.
Female gonopods: densely setose, with 2+2 long and pointed spurs slightly bent
outwards, and a single blunt claw; outer spur 1.4-1.5 times longer than the inner one,
approx. 4 times longer than broad at base; 3-4 dorsal setae on article 1; 12 on article 2.
Plectrotaxy: as in Table 2.
Table 2.
Plectrotaxy of E. cavernicolus Komericki & Stoev sp. n., female paratype.
Ventral Dorsal
Cx Tr Pf F T Cx Tr Pf F T
1 amp amp amp amp a-p a
2 amp amp amp amp a-p a-p
3 amp amp amp amp a-p a-p
4 amp amp amp amp a-p a-p
5 amp amp amp amp a-p a-p
6 amp amp amp amp a-p a-p
7 amp amp amp amp a-p a-p
8 amp amp amp amp a-p a-p
9 amp amp amp amp a-p a-p
10 amp amp amp amp a-p a-p
11 amp amp amp (a) amp a-p a-p
12 m amp amp amp (a) amp a-p a-p
13 m amp amp amp a amp a-p a-p
14 am m amp amp a a amp a-p p
15 am m amp am a a amp p -
18
Stoev P et al.
Description of other female paratypes: Length: 19-22 mm; ocelli: 1+10—1+11;
antennae composed of 65-68 articles; coxosternal teeth: 7+7. Tergites: TT8, 10 and 11
slightly emarginated; posterior margin of TT2, 4, 6, 7 straight. Legs: seriate setae
missing on the tarsi 1 and 2 of leg 15, present in one short row only on posterior part of
tarsus 2. Female gonopods: with 2+2 elongated sharply pointed spurs slightly bent
outwards and a single blunt claw; 3-4 dorsal setae on article 1; 8 on article 2.
Sometimes, a small, pointed spine occurs posteriorly in the middle of the first genital
segment; so far, it has been detected only in two adult females [Kaczmarek (1973)
reported similar spur in Polybothrus ochraceus (Folkmanova, 1936) (= E.
transsylvanicus, cf. Stoev 2002)].
Diagnosis
The species can be readily distinguished from all other congeners by the following set
of molecular and morphological characters: interspecific genetic distance in COI from
the closest neighbour, E. leostygis: 14.5-15.4%; antennae moderately long (approx.
70% body length), comprised of 67-71 articles; 11-15 ocelli; 6+6-8+8 coxosternal teeth;
tergites 9, 11, 13 with posterior triangular projections; intermediate tergite
subpentagonal, posterior margin deeply emarginated, middle part of posterior third of
tergite densely covered with setae; laterally, on both sides of the central setose area,
there are two specific seta-free regions; pretarsus 15 without accessory spines; leg 15
long (approx. 70-75% body length), prefemur of male leg 15 with a large, apically
rounded proximal knob protruding mediad, latter slightly bent dorsad and bearing a
cluster of long setae on tip; distal part of prefemur with a well-defined circular
protuberance covered with setae; posterior margin of male first genital sternite deeply
emarginated, nearly as deep as half of the sternite’s length.
Etymology
Cavernicolus means “living in caves or caverns’, to emphasise that the species
inhabits caves.
Description of the type locality
Eupolybothrus cavernicolus Komericki & Stoev sp.n. is so far Known only from the
caves Miljacka Il and Miljacka IV (= Spilja kod mlina na Miljacki), situated near the
village of Kistanje, Krka National Park, Knin District, Croatia (Fig. 12). The two caves
are situated close to each other and are formed in Middle Eocene to Early Oligocene
conglomerate and marbly limestone. Miljacka II is the longest cave in the Krka National
Park, with a large, spacious entrance and a total length of over 2800 m (Fig. 13). Most
of the cave passages are under water except for approx. 300 m of main passage. From
a hydrogeological point of view, cave Miljacka II contains a periodical spring, while cave
Miljacka IV has a permanent water flow. The cave Miljacka IV has two entrances, one
dry and one underwater, and a length of approximately 43 m. The land entrance is
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 19
walled in and with a small door while inside the cave there is a thick drywall separating
it in two parts. The climatic conditions in Miljacka II as measured on 4" May 2010 and
8'" October 2010 are as follows: Tair = 12.5-13.7°C (Kestrel); RH = 100%; Tsediment =
12.5-13.2°C; Twater = 12.6-13.2°C; in Miljacka IV (measured on 4 May 2010): Tair =
13.1-13.6°C (Kestrel); RH = 100%; Tsediment = 12.5°C; Twater = 12.5°C. In Miljacka Il,
the specimens were collected in the aphotic zone, approx. 50 m from the cave
entrance, in a passage where water never occurs in a periodic flow. In Miljacka IV, they
were found closer to the entrance, under stones.
Figure 12.
Map of Croatia showing the locality of Eupolybothrus cavernicolus Komericki & Stoev sp. n.
Figure 13.
Entrance of cave Miljacka Il, type locality of Eupolybothrus cavernicolus Komericki & Stoev
sp. n.
20
Stoev P et al.
Associated fauna: Gastropoda: Oxychilus cellarius (O.F. Muller, 1774), Hauffenia
Jadertina Kuscer, 1933, Hadziella sketi Bole, 1961; Araneae: Episinus cavernicola
(Kulczynski, 1897), Nesticus eremita Simon, 1879, Tegenaria domestica (Clerck,
1757), Metellina merianae (Scopoli, 1763), Histopona sp.; Pseudoscorpiones:
Chthonius tetrachelatus (Preyssler, 1790), Chthonius litoralis Hadzi, 1933, Neobisium
carsicum Hadzi, 1933, Pselaphochernes litoralis Beier, 1956; Opiliones: Nelima
troglodytes Roewer, 1910; Acari: Parasitus sp.; |sopoda: Monolistra pretner Sket,
1964, Sphaeromides virei mediodalmatina Sket, 1964, Alpioniscus balthasari
(Frankenberger, 1937), Cyphopleon kratochvili (Frankenberger, 1939); Amphipoda:
Niphargus sp.; Decapoda: Troglocaris sp.; Chilopoda: Eupolybothrus tridentinus,
Harpolithobius sp., _Lithobius sp., Cryptops sp.; Diplopoda: Brachydesmus
subterraneus Heller, 1858; Collembola: Troglopedetes pallidus Absolon, 1907,
Heteromurus nitidus (Templeton, 1835), Pseudosinella heteromurina (Stach, 1929),
Lepidocyrtus sp.; Diplura: Plusiocampa (Stygiocampa) dalmatica Conde, 1959,
Japygidae gen. spp.; Coleoptera: Laemostenus cavicola muilleri (Schaum, 1860),
Atheta spelaea (Erichson, 1839); Orthoptera: Dolichopoda araneiformis (Burmeister,
1838), Troglophilus ovuliformis Karny, 1907, Gryllomorpha dalmatina Ocskay, 1832;
Psocoptera: Psyllipsocus sp.; Lepidoptera: Apopestes spectrum (Esper, 1787);
Amphibia: Proteus anguinus Laurenti, 1768; Chiroptera: a colony of bats, Myotis
capaccinii (Bonaparte, 1837) (Margus et al. 2012).
Eupolybothrus leostygis (Verhoeff, 1899)
BOLD dataset http://dx.doi.org/10.5883/DS-EUPCAV
Plazi http://plazi.org:8080/GgSRS/html? CCEB9C62C87766E980DD858BC13468C8
GenBank KF 715047
GenBank KF 715051
GenBank KF 715053
GenBank KF 715060
GenBank KF 715055
GenBank KF 715056
GenBank KF 715057
GenBank KF 715058
MorphBank 999021822&tsn=true
Nomenclature
Lithobius (Polybothrus) leostygis Verhoeff, 1899 - Verhoeff 1899: 451-452.
Material
Other material:
a. country: Croatia; stateProvince: Dubrovnik-Neretva; locality: Jama u Zabiradu, Zabirade,
Osojnik; samplingProtocol: hand collected; eventDate: 30 March 2008; individualCount: 1;
sex: male; lifeStage: adult; recordedBy: J. Bedek; institutionCode: ZMUC; collectionCode:
zmuc00029441
Eupolybothrus cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 21
Notes
As the morphology of E. /eostygis is still insufficiently known, we provide here scanning
electron microscope images (Figs 14, 15, 16) based on an adult male specimen
collected in Jama u Zabiradu Cave.
Figure 14.
Eupolybothrus leostygis (Verhoeff, 1899), male.
a: ocelli
b: forcipules, ventral view
Figure 15.
Eupolybothrus leostygis (Verhoeff, 1899), male.
a: tergite 14 and intermediate tergite, dorsal view
b: close up of posterior part of prefemur of leg 14 showing the expanded distal part bearing
feebly defined setose protuberance
22 Stoev P et al.
Identification keys
Identification key to the species of Eupolybothrus (Schizopolybothrus)
based on adult males
—_
Six poorly defined, feebly pigmented ocelli [original description] E. leostygis
— 10-25 pigmented ocelli 2
2 Leg 15 with a large knob on prefemur (Figs 8b, 16) 3
— Leg 15 without such knob (Fig. 17a) [original description] E. tabularum
Figure 16.
Eupolybothrus leostygis (Verhoeff, 1899), male: prefemur 15 showing the bare knob, dorsal
view.
Figure 17.
Prefemur of male leg 15. From Stoev et al. (2010).
a: Eupolybothrus tabularum
b: Eupolybothrus excellens
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ...
Prefemoral knob apically incised forming two rounded and
densely setose processes (Fig. 17b) [original description]
Prefemoral knob simple (Fig. 16)
Prefemoral knob with a cluster of setae (Fig. 8a)
Prefemoral knob without such cluster of setae (Fig. 16)
Antennae with 50-60 antennal articles
Antennae with 70-83 antennal articles
Prefemoral knob poorly developed (Fig. 18a) [original
description]
Prefemoral knob large (Fig. 18b) [original description]
E. excellens
4
E. cavernicolus
sp. n.
5
6
7
E. caesar
E. spiniger
Figure 18.
Prefemur of male leg 15. From Stoev et al. (2010).
a: Eupolybothrus caesar
b: Eupolybothrus spiniger
Coxosternal teeth: 10+10-11+11; 1 ventral spine on tibia of leg
15 [original description]
Coxosternal teeth: 8+8-9+9; 2 ventral spines on the tibia of leg
15
Antennae with 74 antennal articles, ocelli 1+14 [original
description]
Antennae with more than 81-83 articles, ocelli 1+18-1+19 [
original description]
E. stygis
E. acherontis
E. a.
wardaranus
23
24 Stoev P et al.
Analysis
Molecular delimitations
The ABGD approach clustered the 37 Eupolybothrus specimens into 12 groups (Fig. 19).
Ten of them agreed with morphospecies designations: E. grossipes, E. leostygis, E. kahfi,
E. litoralis, E. nudicornis, E. fasciatus, E. transsylvanicus, E. obrovensis, E. gloriastygis
and E. cavernicolus Komericki & Stoev sp. n. The remaining two genetic clusters are each
formed by a specimen of the morphospecies E. tridentinus from Germany. The reversed
SP networks support most of the ABGD results and morphospecies assignments, but split
both E. /eostygis and E. tridentinus in two and E. nudicornis in three clusters, respectively.
We follow a conservative approach here and refer to the ABGD results, which largely
correspond to morphology, E. tridentinus being the only exception which could suggest a
case of cryptic diversity and will require further investigation. All delineated groups have a
bootstrap support of 100 in the NJ-tree topology. The interspecific K2P genetic distance
ranges from 10.7 % for the species pair E. tridentinus (GER1) — E. tridentinus (GER2) to
24.5 % for E. grossipes — E. cavernicolus Komericki & Stoev sp. n. (Table 3). Intraspecific
K2P genetic distance is maximal for E. nudicornis (7.2 %) and minimal for individuals of E.
obrovensis and E. gloriastygis and the species E. cavernicolus Komericki & Stoev sp. n.
(0.0 %) (Fig. 19).
Table 3.
Interspecific genetic distances (K2P) of Eupolybothrus species. Given are the ranges from
minimum to maximum values.
1. €£ gloriastygis
BOLD:AAY5019
2 E. leostygis 16.7 -
BOLD:AAY5071 17.8
3. E. obrovensis 16.2- 18.5-
BOLD:AAY5641 17.0 19.4
4 E. cavernicolus 176- 14.5- 20.8-
BOLD: AAY4900 18.0 154 21.2
5 E litoralis 14.7- 17.1- 17.1- 18.0 -
151 17.5 17.3 18.1
6 E._ fasciatus 16.3- 18.7- 17.5- 21.9- 13.7
16.8 19.2 17.7 22.1
7 €E. tridentinus GER1 17.7- 16.7- 18.3- 17.4- 18 18.3
BOLD:AAV7132 18.0 17.3 185 17.7
8 E. tridentinus GER2 17.4- 186- 19.4- 18.1- 15.7 17.5 10.7
BOLD:AAV7131 17.8 191 19.7 18.4
9 E. transsylvanicus 20.4- 20.7- 21.4- 206- 16.0- 20.4- 181 19.7-
BOLD:AAJ0488 21.3 21.6 22.1 20.7 16.4 20.8 20.1
10. E. kahfi 21.9- 18.9- 21.6- 20.0- 21 21.7 22.3 21.5 23.2-
BOLD:AAY2955 22.5 20.1 21.8 20.2 23.6
Eupolybothrus cavernicolus Komericki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 25
11 E. nudicornis 20.1- 19.4- 21.1- 21.2- 20.1- 21.7- 20.7- 19.4- 21.4- 17.2 -
BOLD:AAN2808 23.2 21.8 241 22.7 21.7 226 22.4 21.0 223 18.8
BOLD:AAN2810
BOLD:AAN2811
12 E. grossipes 19.2- 21.0- 20.9- 24.2- 166 153 209 189 20.3 22.1 20.7-
BOLD:AAY7960 196 21.9 21.1 24.5 22.1
SP ABGD @ intra-K2P
(min-max)
EUCRO16-11
0.7 %
4, E. leostygis
too 7 EUGROIS-11 (0-2.1 %) yg!
— EUCROS4-11
100° EUCRO44-11
~ EUCRO42-11
EUCRO43-11
EUCRO41-11
0.0% E. cavernicolus
CHIPSO17-09
4997 CHIPSI74-10
— GHIPS173-10
CHIPS 170-09
CHIPS 171-09
E. kahfi
4% 7
o- r) 2 %) E. nudicornis
MYFAB431-11
MYFAB430-11
EUCROS0-11
E. tridentinus (GER1)
E. tridentinus (GER2)
E. grossipes
E. fasciatus
E. litoralis
AY214420
CHIPS049-10
100°CHIP'S052-10
CHIPS053-10
0.3% — E, transsylvanicus
EUCRO27-11
100) EUCRO26-11
ene ) E. gloriastygis
100, EUCRO23-11
EUCRO21-11
EUCRO22-11
= MYFAB442-11 L. austriacus
MYFAB443-11 rT
ae L. erassipes
19 ;
‘or 2%) E. obrovensis
Figure 19.
Delineation of Eupolybothrus species — Neighbor joining tree K2P distances. Visualised are the
clusters obtained from the reversed Statistical Parsimony (SP) method and the Automatic
Barcoding Gap Discovery (ABGD) procedure. Bootstrap support for the identified lineages are
given above. The intraspecific genetic variability is given for each cluster. Source data is available
in Suppl. material 1.
Transcriptome analysis and annotation
The raw data was first filtered by removing inadequate reads with: 1) adapter
contamination; 2) >10 Ns; 3) >50 base pairs of low quality (quality value <65). The resulting
2 Gb of clean data were processed into subsequent assemblies using SOAPdenovo_trans
(Xie et al. 2013) under default parameters. The abundance information was provided
directly by SOAPdenovo_trans, and played no roles in the subsequent analysis steps. A
total of 67,785 scaffolds were produced with an average length of 812 bp and N50 of 1,448
bp [see GigaDB (Stoev et al. 2013)]. Subsequent annotation was conducted by tracing
homologs against currently available databases, including Nr, SwissProt and COG. Using
this method, 22,866 scaffolds were functionally annotated (Fig. 20a, b, c). Annotated genes
were then translated to peptide sequences via CDS prediction according to their blast
results using GeneWise (Birney et al. 2004) (see GigaDB (Stoev et al. 2013)). Using
orthoDB (http://cegg.unige.ch/orthodb6) (Waterhouse et al. 2012), 2,188 one to one
orthologs were filtered out from four selected arthropod genomes: Drosophila
melanogaster Meigen, 1830, Daphnia pulex (Linnaeus, 1758), Ixodes scapularis Say, 1821
and Strigamia maritima (Leach, 1817). HaMstR (Ebersberger et al. 2009) was applied to
search corresponding orthologous genes in our transcriptome data, delivering 1,668
26 Stoev P et al.
predicted orthologs of both nucleotide and protein sequences (see GigaDB (Stoev et al.
2013)).
(1) Evalue Distribution (10) Similarity Gistributior
a CC ————— b
famine ool Linge
od 2172") =o r.->
15 2. - oe oe
ne)
5.7 DO,
Figure 20.
Gene annotation. Original data available from GigaScience GigaDB (Stoev et al. 2013).
a: E-value, identity and species distribution statistics of the sequences that can find homologs on
Nr database
b: COG functional classification of the transcripts
c: GO categories of the transcripts
Discussion
Taxonomic affinities
According to the division of the subgenera of Eupolybothrus of Jeekel (1967), E.
cavernicolus Komericki & Stoev sp.n. falls into subgenus Schizopolybothrus Verhoef,
1934, characterized by the presence of triangular projections on tergites 9, 11, 13, a VCm
spine on leg 15, one or more VCa spines and a single claw on the pretarsus of leg 15. The
same author further distinguishes three species groups in the subgenus based on the
morphology of male gonopods and presence/absence of modifications on leg 15:
* Group |, characterized by short male gonopods and presence of a large knob on male
prefemur 15, currently including E. caesar (Verhoeff, 1899), from Bosnia-Herzegovina,
Albania, mainland Greece (incl. lonian Is.) and Macedonia (FYROM); E. spiniger
(Latzel, 1888), from Bosnia-Herzegovina; E. acherontis (Verhoeff, 1900), from Bosnia-
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 27
Herzegovina (E. a. acherontis) and Macedonia (FYROM) (E. a. wardaranus (Verhoeff,
1937)); E. stygis (Folkmanova, 1940), from Bosnia-Herzegovina; and E. /eostygis, from
Croatia and Bosnia-Herzegovina (see Kos 1992, Stoev 1997, Zapparoli 2002). Here
also belongs a new cave-dwelling species from Velebit, Croatia, recently discovered by
AK and the CBSS team, whose description is currently in progress. While E. caesar
and E. leostygis have recently been validated and re-described (see Eason 1983,
Zapparoli 1984, Zapparoli 1994), the status of the other four taxa remains uncertain
(see e.g., Stoev 2000, Stoev 2001, Stoev et al. 2010)
Group Il, lacking any specific modifications on male legs while gonopods are also
short, encompassing E. tabularum (Verhoeff, 1937) from the Western Alps and E.
excellens (Silvestri, 1894) from the Ligurian Apennines.
Group III, characterized by the long gonopods and dorsal furrow on male prefemur 15,
with E. zeus (Verhoeff, 1901) from Central Greece and E. sissii (Kanellis, 1959) from
Euboea Island, Greece. Both species are currently considered junior synonyms of the
widespread Carpathian-Balkan species Eupolybothrus (Mesobothrus) transsylvanicus
(cf. Zapparoli 1994).
Jeekel’s division of the genus (Jeekel 1967) is quite artificial and does not reflect real
evolutionary relationships as it is merely based on a few morphological traits. Some
species were certainly misplaced in these groupings, as for example E. excellens, of
which, males show noticeable modifications on leg 15 (see Fig. 17b). Two other species, E.
zeus and E. sissii, were even excluded from Schizopolybothrus (cf. Zapparoli 1994,
Zapparoli 2002). Showing a prominent prefemoral knob on male leg 15 and having
relatively short gonopods, E. cavernicolus Komericki & Stoev sp.n. unquestionably
belongs in Group I, as defined by Jeekel (1967). The new species can be readily
distinguished from other members of Eupolybothrus (Schizopolybothrus) by the presence
of a large proximal knob surmounted by a characteristic cluster of setae, and distal setose
protuberance of male prefemur 15. In addition, the species presents a different
arrangement of spiniform setae on the intermediate tergite.
Micro-computed tomography and ‘cybertype’ notion
The new generation imaging technologies, such as magnetic resonance imaging (MRI) and
micro-computed tomography (micro-CT) are opening new horizons in biology (Mietchen et
al. 2008, Ziegler et al. 2008). Micro-CT is becoming widely used in comparative,
developmental and functional biology (see e.g., Metscher 2009a, Metscher 2009b,
Wojcieszek et al. 2012), paleontology (Blazejowski et al. 2011, Edgecombe et al. 2012),
molecular biology (Metscher and Muller 2011) and taxonomy (Faulwetter et al. 2013,
Michalik et al. 2013). By employing micro-CT scans in taxonomy, important morphological
and anatomical characters can be examined in their natural position without damage to the
original specimen. This allows researchers to re-assess character shape and functionality
or even discover new diagnostic characters (Ziegler et al. 2010, Zimmermann et al. 2011,
Faulwetter et al. 2013). To make type material continuously and simultaneously available to
taxonomists and to improve access to high-quality morphological data, Faulwetter et al.
(2013) suggested the creation of high-resolution virtual morphological and anatomical data
28 Stoev P et al.
libraries allowing reconstruction and interactive manipulation of type specimens, the so-
called ‘cybertypes’.
The ‘cybertype’ notion is herewith tested for the first time with the newly described taxon
(Fig. 21), for which a rich image library has been created to allow its subsequent
recognition, virtual manipulation and reuse. This image library, from which the 3D model is
created, has been deposited in the GigaScience database, GigaDB, as a zip and a gzipped
tar archive containing BMP images (Stoev et al. 2013). The 3D model was converted into
an AVI file, using the flight recorder of CTVox, and disseminated, along with the video of
the living specimen (Fig. 22) through YouTube. According to Faulwetter et al. (2013), a
‘cybertype’ should be linked to the original type material and be retrievable and freely
accessible. We comply with these requirements by a) including a set of Darwin Core files
along with the deposited volumetric data which describe the attributes and deposition of
the physical type material and b) using a CCZero license and rich metadata to make the
"cybertype" retrievable and reusable. Furthermore, through the same set of Darwin Core
files, the morphological data are also linked to the transcriptomic data at GigaDB,
effectively extending the ‘cybertype’ concept and providing direct links to other data
describing type material of the same species.
Figure 21.
Eupolybothrus cavernicolus Komericki & Stoev sp.n., paratype, 3D model, volume rendering,
created with CT Vox, virtual rotation and dissection. Movie available at: YouTube
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 29
Figure 22.
Movie of Eupolybothrus cavernicolus Komericki & Stoev sp. n., holotype, filmed ex-situ in a plastic
container. Movie available at: YouTube
Data management and release
Whereas a lack of reference genomes in non-model organisms has hampered genetic and
phylogenomic studies, transcriptomes may present a time and cost-effective substitute to
whole genome sequencing for these types of studies and an efficient way to produce
massive amounts of gene sequence data. While transcriptomic studies of centipede
species, e.g. Alipes grandidieri Lucas, 1864 (Chilopoda; Scolopendromorpha), exist in the
literature (Riesgo et al. 2012), centipede genome data in public and accessible repositories
are still scarce and difficult to find. To address this deficiency, and to produce a model of an
accessible resource for the community, all of the transcriptomic data have been made
available under the highest metadata standards, both in relevant community specific
databases (raw data in the SRA [SRA project accession: ERP003841] and transcriptomic
in ArrayExpress [accession E-MTAB-1859]), as well as GigaDB (Stoev et al. 2013).
GigaDB collects together all of the genomic and morphological data, and utilises the large
computing infrastructure of the BGI and Aspera data transfer capabilities, able to host and
deliver much larger and heterogeneous datasets than other repositories (Sneddon et al.
2012). Datasets are also issued with DOls, which are discoverable through the DataCite
metadata search engine and Thomson Reuters Data Citation Index, and can be integrated
into a publication or independently cited.
In addition to making data publicly available, it is crucial to provide rich metadata to enable
data interoperability and reuse. As there is only one transcriptome available, it is not
possible to include additional ‘factor’ information. However, by including sequence reads,
experimental design, protocols and processed data we were able to produce the maximum
amount of (4*) MINSEQE compliant metadata (Brazma 2009). To maximise its
interoperability, the metadata are also available from GigaDB in ISA-TAB format (Sansone
et al. 2012).
30 Stoev P et al.
For volumetric data created by techniques such as micro-CT and micro-MRI, no
community standards exist yet. The DICOM standard (Digital Imaging and
Communications in Medicine, http://dicom.nema.org/) used by the medical community is
not tailored for taxonomic purposes, thus its usefulness for this research field still has to be
investigated (Faulwetter et al. 2013). However, even in the absence of widely accepted
standards, we provide rich metadata for the micro-CT data, based on the metadata
descriptors at Morphosource (http://morphosource.org). The same set of descriptors has
been used by GigaDB, where we also applied the ISA-TAB format in order to ensure re-
usability and interoperability of the data (Sansone et al. 2012), describing all parameters
and settings used to create the data. The data package of micro-CT deposited at GigaDB
thus contain:
* MicroCT image stack available in 2 different formats:
° Several ZIP files, each contains 500 bmp images, the scanning log documentation
file and Darwin Core type specimen data.
° A single gzipped TAR archive of all 3876 bmp images, the scanning log
documentation file and Darwin Core type specimen data.
* Documentation of the scanning and reconstruction process through ISA-TAB metadata
provided by GigaDB and the inclusion of the scanning log file with the ‘cybertype’.
* Specimen data in Darwin Core format and link to the location of the physical material
and the transcritomic data through Darwin Core comma-separated value format (CSV)
files:
° Eupolybothrus_cavernicolus_sequenced_vaucher_ paratype.csv
° Eupolybothrus_cavernicolus_micro-CT_vaucher_ paratype.csv
° Eupolybothrus_cavernicolus_all_types.csv
* ISA-TAB metadata that ensure retrievability and interoperability.
In combination with the Darwin Core files describing the specimen data, we thus fully
annotate and document the ‘cybertype’ of Eupolybothrus cavernicolus Komericki & Stoev
sp. n. The generation of large molecular and morphological data pools that originate from
type specimens increases the applicability of taxonomic data in other scientific disciplines
such as comparative morphology, evolutionary biology, medicine, ecology. The new holistic
approach raises important questions and shows up new directions for developments of
biodiversity data management about the lack of mechanisms for cross-linking molecular
and morphological data and global metadata standards for micro-CT and transcriptomic
data, as well as absence of reliable data repositories for micro-CT image libraries.
Also, as a pilot project, we annotate all currently valid Eupolybothrus (Schizopolybothrus)
species with their original descriptions that were extracted from the original publications
through applying optical character recognition (OCR) and additionally tagged by using
Golden Gate software (Sautter et al. 2007). All species treatments are deposited at Plazi.
This represents part of a more ambitious project aiming at digitization of all species
descriptions and important taxonomic treatments of Eupolybothrus species that is currently
being carried out in the framework of the pro-iBiosphere project (http://www.pro-
ibiosphere.eu).
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 31
To create reliable links between the published sequence IDs and BOLD, an online dataset
DS-EUPCAV was generated in the BOLD system, through which the respective Barcode
Index Numbers (BINs) of the specimens barcoded for this study may be tracked (for the
BIN concept see Ratnasingham and Hebert 2013). All COl sequences were registered in
GenBank, following a newly launched metadata standard in the GenBank taxonomy
database that flags sequences of type specimens.
Conclusions
This study demonstrates a holistic approach to the description of a new taxon, extending
the conventional written description and two-dimensional illustrations with an array of
different information types. While this novel approach contributes to the different aspects of
the species' identity, its main aim is to provide an integrated approach to handling and
publishing large data sets associated with a taxon. The generation of large molecular and
morphological data pools that originate from type specimens increases the applicability of
taxonomic data in other scientific disciplines such as comparative morphology, evolutionary
biology, medicine, ecology, and others.
The concept of a “cybertype” is discussed in the study, but at the same time new questions
arise, pertaining to the definition of such a “cybertype” and they will have to be addressed
by the taxonomy community. Several different kinds of data belonging to the “cybertype”
concept are treated in this study, from free text to sequence data, and from images to
volumetric data. Questions have to be addressed such as whether a cybertype should only
be restricted to morphological data, what data can be used to constitute a cybertype and
whether a cybertype can be composite (i.e. consisting of several data types) or even
distributed (different parts of the data residing on different physical servers). Further
problems to be addressed are the lack of appropriate mechanisms for cross-linking
molecular and morphological data, as well as the absence of global metadata standards
and reliable data repositories for micro-CT image libraries. The metadata descriptors for
micro-CT files used by the Morphosource and GigaDB repositories are a good starting
point for that, as is the use of ISA-TAB to integrate everything together. Whatever the
answers to these questions, there is one mandatory requirement for data that we can
already identify: discoverability and accessibility.
With complex taxon descriptions such as the present one, we are entering new dimensions
of data volumes that have to be managed properly to realise their true value. The
deposition of large data pools in appropriate repositories is not yet straightforward, and
such initiatives have started to emerge only recently. It is our task to ensure from the
beginning that they do not develop into isolated data worlds but that they support
community standards, describing the datasets in a way that they can be retrieved and
cross-linked. Currently, even in modern taxon descriptions, different pieces of data are only
linked through a central locus: the published article. In a future, data-centric world of
taxonomy, articles published through next generation journal workflows will become an
even more important node in a linked network of data elements describing the taxon.
These data elements have to be defined and made accessible through persistent
32 Stoev P et al.
identifiers — not unlike the traditional practice for physical specimens that are accessible
through their museum accession number. In combination with rich metadata standards,
taxonomy will thus open itself up to the semantic Web with its possibilities for intelligent,
complex queries.
In this study, we have taken a first step towards that direction. All data have been
deposited in publicly accessible repositories, such as GigaDB, NCBI, BOLD, Morphbank
and Morphosource, and the respective open licenses used ensure their accessibility and
re-usability. GigaDB in this example provides direct links between the genomic and micro-
CT data, through a Darwin Core CSV dataset describing the type specimens, as well as
capturing all of the metadata in the interoperable ISA-TAB format. Molecular data and
images are annotated with rich metadata to ensure discoverability and reuse. Techniques
such as micro-CT are, however, still in their infancy, and no standardised metadata
schemas exist yet — a gap that needs urgently to be addressed by the community if we are
to avoid a proliferation of isolated datasets.
Taxonomy is at a turning point in its history. New technologies allow for creation of new
types of information at high speed and in gigantic volumes, but without clear rules for
communication standards, we will not be able to exploit their full potential. We need to
focus our efforts on linking these bits and pieces together, by documenting them, by
standardising them and by making them retrievable. If such an infrastructure is in place,
unforeseen analytical powers can be unleashed upon these data, creating a revolution in
our abilities to understand and model the biosphere.
Acknowledgements
This project was developed in collaboration between several research institutions and
driven by Pensoft Publishers, BGl-Shenzhen and GigaScience. We would like to thank
Philippe Rocca-Serra and the ISA-Team for help in producing the ISA-TAB metadata. We
are very grateful to Biodiversity Data Journal editor Bob Mesibov (Queen Victoria Museum
and Art Gallery, Tasmania, Australia), and the referee Greg Edgecombe (NHM, London) for
their constructive comments and useful suggestions that greatly improved the manuscript.
Special thanks to Henrik Enghoff who facilitated AK and PS’ respective visits to the Natural
History Museum of Denmark, financially supported by the European Commission’s (FP 6)
Integrated Infrastructure Program SYNTHESYS (DK-TAF). All specimens were collected
during cave fauna research projects conducted by the Croatian Biospeleological Society
and funded by The Krka National Park. AK thanks all colleagues from the CBSS who
assisted her in collecting the specimens. Stylianos Simaiakis (Natural History Museum of
Crete) kindly provided material from the type locality of E. /itoralis for DNA barcoding.
Pensoft has received financial support by the EU FP7 projects VIBRANT (Virtual
Biodiversity Research and Access Network for Taxonomy, www.vbrant.eu, Contract no.
RI-261532) and pro-iBiosphere (Coordination & Policy Development in Preparation for a
European Open Biodiversity Knowledge Management System, Addressing Acquisition,
Curation, Synthesis, Interoperability & Dissemination, Contract no. RI-312848, www.pro-
Eupolybothrus cavernicolus Komericéki & Stoev sp. n. (Chilopoda: Lithobiomorpha: ... 33
ibiosphere.eu). The BGI and GigaScience teams have received support from China
National Genebank (CNGB). The DNA barcodes were obtained through the International
Barcode of Life Project supported by grants from NSERC and from the government of
Canada through Genome Canada and the Ontario Genomics Institute.
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Supplementary material
Suppl. material 1: Raw data used for COI delineation of the Eupolybothrus species
Authors: Stoev et al. 2013
Data type: genomic
Brief description: The archive contains the following data: 1) fasta-Alignment as the basis for all
analyses (.FASTA), 2) mega-file for the calculation of the genetic distances and the NJ tree
(.MDSX), 3) NJ-tree in Newick format (.NWkK), 4) graph of the TCS Software for the Statistical
Parsimony method (.GRAPH)
Filename: E_cavernicolus.rar - Download file (10.35 kb)
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